Suppression of specific classes of soybean seed protein genes

ABSTRACT

This invention concerns the construction of transgenic soybean lines wherein the expression of genes encoding seed storage proteins are modulated to effect a change in seed storage protein profile of transgenic plants. Modification of the seed storage protein profile can result in the production of novel soy protein products with unique and valuable functional characteristics.

FIELD OF THE INVENTION

[0001] This invention concerns the construction of transgenic soybeanlines wherein the expression of genes encoding seed storage proteins ismodified to effect a change in seed storage protein profile oftransgenic plants. Such modified transgenic soybean lines are used forthe production of novel soy protein products with unique and valuablefunctional characteristics.

BACKGROUND OF THE INVENTION

[0002] Soybean seeds contain from 35% to 55% protein on a dry weightbasis. The majority of this protein is storage protein, which ishydrolyzed during germination to provide energy and metabolicintermediates needed by the developing seedling. The soybean seed'sstorage protein is an important nutritional source when harvested andutilized as a livestock feed. In addition, it is now generallyrecognized that soybeans are the most economical source of protein forhuman consumption. Soy protein or protein isolates are already usedextensively for food products in different parts of the world. Mucheffort has been devoted to improving the quantity and quality of thestorage protein in soybean seeds.

[0003] The seeds of most plant species contain what are known in the artas seed storage proteins. These have been classified on the basis oftheir size and solubility (Higgins, T. J. (1984) Ann. Rev. PlantPhysiol. 35:191-221). While not every class is found in every species,the seeds of most plant species contain proteins from more than oneclass. Proteins within a particular solubility or size class aregenerally more structurally related to members of the same class inother species than to members of a different class within the samespecies. In many species, the seed proteins of a given class are oftenencoded by multigene families, sometimes of such complexity that thefamilies can be divided into subclasses based on sequence homology.

[0004] There are two major soybean seed storage proteins:glycinin (alsoknown as the 11S globulins) and β-conglycinin (also known as the 7Sglobulins). Together, they comprise 70 to 80% of the seed's totalprotein, or 25 to 35% of the seed's dry weight. Glycinin is a largeprotein with a molecular weight of about 360 kDa. It is a hexamercomposed of the various combinations of five major isoforms (commonlycalled subunits) identified as G1, G2, G3, G4 and G5. Each subunit is inturn composed of one acidic and one basic polypeptide held together by adisulfide bond. Both the acidic and basic polypeptides of a singlesubunit are coded for by a single gene. Hence, there are fivenon-allelic genes that code for the five glycinin subunits. These genesare designated Gy1, Gy2, Gy3, Gy4 and Gy5, corresponding to subunits G1,G2, G3, G4 and G5, respectively (Nielsen, N. C. et al. (1989) Plant Cell1:313-328).

[0005] Genomic clones and cDNA's for glycinin subunit genes have beensequenced and fall into two groups based on nucleotide and amino acidsequence similarity. Group I consists of Gy1, Gy2, and Gy3, whereasGroup II consists of Gy4 and Gy5. There is greater than 85% similaritybetween genes within a group (i.e., at least 85% of the nucleotides ofGy1, Gy2 and Gy3 are identical, and at least 85% of the nucleotides ofGy4 and Gy5 are identical), but only 42% to 46% similarity between thegenes of Group I and Group II.

[0006] β-Conglycinin (a 7S globulin) is a heterogeneous glycoproteinwith a molecular weight ranging from 150 and 240 kDa. It is composed ofvarying combinations of three highly negatively charged subunitsidentified as α, α′ and β. cDNA clones representing the coding regionsof the genes encoding the the α and α′ subunits have been sequenced andare of similar size; sequence identity is limited to 85%. The sequenceof the cDNA representing the coding region of the β subunit, however, isnearly 0.5 kb smaller than the α and α′ cDNAs. Excluding this deletion,sequence identity to the α and α′ subunits is 75-80%. The three classesof β-conglycinin subunits are encoded by a total of 15 subunit genesclustered in several regions within the genome soybean (Harada, J. J. etal. (1989) Plant Cell 1:415-425).

[0007] New soy based products such as protein concentrates, isolates,and textured protein products are increasingly utilized in countriesthat do not necessarily accept traditional oriental soy based foods. Useof these new products in food applications, however, depends on localtastes and functional characteristic of the protein products relative torecipe requirements. Over the past 10 years, significant effort has beenaimed at understanding the functional characteristics of soybeanproteins. Examples of functional characteristics include water sorptionparameters, wettability, swelling, water holding, solubility,thickening, viscosity, coagulation, gelation characteristics andemulsification properties. A large portion of this body of research hasfocused on study of the β-conglycinin and glycinin proteinsindividually, as well as how each of these proteins influences the soyprotein system as a whole (Kinsella, J. E. et al. (1985) New ProteinFoods 5:107-179; Morr, C. V. (1987) JAOCS 67:265-271; Peng, L. C. et al.(1984) Cereal Chem 61:480-489). Because functional properties aredirectly related to physiochemical properties of proteins, thestructural differences of β-conglycinin and glycinin result in these twoproteins having significantly different functional characteristics.Differences in thermal aggregation, emulsifying properties, and waterholding capacity have been reported. In addition, gelling propertiesvary as well, with glycinin forming gels that have greater tensilestrain, stress, and shear strength, better solvent holding capacity, andlower turbidity. However, soy protein products produced today are ablend of both glycinin and β-conglycinin and therefore have functionalcharacteristics dependent on the blend of glycinin's and β-conglycinin'sindividual characteristics. For example, when glycinin is heated to 100°C., about 50% of the protein is rapidly converted into solubleaggregates. Further heating results in the enlargement of the aggregatesand in their precipitation. The precipitate consists of the glycinin'sbasic polypeptides; the acidic polypeptides remain soluble. The presenceof β-conglycinin inhibits the precipitation of the basic polypeptides byforming soluble complexes with them. Whether heat denaturation isdesireable or not depends on the intended use. If one could produce soyprotein products containing just one or the other storage protein,products requiring specific physical characteristics derived fromparticular soy proteins would become available or would be moreeconomical to produce.

[0008] Over the past 20 years, soybean lines lacking one or more of thevarious storage protein subunits (null mutations) have been identifiedin the soybean germplasm or produced using mutational breedingtechniques. Breeding efforts to combine mutational events have resultedin soybean lines whose seeds contain about half the normal amount ofβ-conglycinin (Takashashi, K. et al. (1994) Breeding Science 44:65-66;Kitamura, J. (1995) JARQ 29:1-8). The reduction of β-conglycinin iscontrolled by three independent recessive mutations. Recombiningglycinin subunit null mutations have resulted in lines whose seeds havesignificantly reduced amounts of glycinin (Kitamura, J. (1995) JARQ29:1-8). Again, reduction is controlled by three independent recessivemutations. Developing agronomically viable soybean varieties from theabove lines, in which the seed contains only glycinin or β-conglycinin,will be time consuming and costly. Each cross will result in theindependent segregation of the three mutational events. In addition,each mutational event will need to be in the homozygous state.Development of high yielding agronomically superior soybean lines willrequire the screening and analysis of a large number of progeny overnumerous generations.

[0009] Antisense technology has been used to reduce specific storageproteins in seeds. In Brassica napus, napin (a 2S albumin) andcruciferin (an 11S globulin) are the two major storage proteins,comprising about 25% and 60% of the total seeds protein, respectively.Napin proteins are coded for by a large multi-gene family of up to 16genes; several cDNA and genomic clones have been sequenced (Josefsson,L.-G. et al. (1987) J. Biol Chem 262:12196-12201; Schofield, S. andCrouch, M. L. (1987) J. Biol. Chem. 262:12202-12208). The genes exhibitgreater than 90% sequence identity in both their coding and flankingregions. The cruciferin gene family is equally complex, comprising 3subfamilies with a total of 8 genes (Rodin, J. et al. (1992) Plant Mol.Biol. 20:559-563). Kohno-Murase et al. ((1994) Plant Mol. Biol.26:1115-1124) demonstrated that a napin antisense gene using the napAgene driven by the napA promoter could be used to construct transgenicplants whose seeds contained little or no napin.

[0010] The same group (Kohno-Murase et al. (1995) Theoret. AppliedGenetics 91:627-631) attempted to reduce cruciferin (11S globulin)expression in Brassica napus by expressing an antisense form of acruciferin gene (cruA, encoding an alpha 2/3 isoform) under the controlof the napA promoter. In this case the results were more complex. Thecruciferins are divided into three subclasses based on sequence identity(alpha 1, 2/3, and 4); the classes each have from 60-75% sequenceidentity with each other (Rodin, J. et al. (1992) Plant Mol. Biol.20:559-563). Expression of the antisense gene encoding the alpha 2/3isoform resulted in lower levels of the alpha 1 and 2/3 forms. However,there was no reduction in the expression of the alpha 4 class.

[0011] Antisense technology was used to reduce the level of the seedstorage protein, glutelin, in rice. Expression of the seed specificglutelin promoter operably linked to the full length antisense glutelincoding region resulted in about a 25% reduction in glutelin proteinlevels (U.S. Pat. No. 5,516,668).

SUMMARY OF THE INVENTION

[0012] The instant invention provides a method for reducing the quantityglycinin or β-conglycinin (11S or 7S globulins, respectively) seedstorage proteins in soybeans. In one embodiment, cosuppressiontechnology was used to suppress the expression of genes encoding the7S-globulin class of seed protein genes. Genes encoding either two (αand α′) or all three subclasses (α, α′ and β) of 7S globulins weresuppressed by expression of the gene encoding a single subclass (α) ofβ-conglycinin, resulting in soybean lines with altered seed storageprofiles. In another embodiment, a method for supressing two completelydifferent genes, only one of which is a seed protein gene, is presented,allowing for multiple changes in seed composition. Surprisingly,expression of a chimeric gene comprising the promoter region of asoybean seed storage protein operably linked to the coding region of asoybean gene whose expression alters the fatty acid profile oftransgenic soybean seeds resulted in simultaneous alteration of twodistinct phenotypic traits: seed storage protein profile and seed oilprofile.

[0013] The method for reducing the quantity of soybean seed storageprotein taught herein comprises the following steps:

[0014] (a) constructing a chimeric gene comprising (i) a nucleic acidfragment encoding a promoter that is functional in the cells of soybeanseeds, (ii) a nucleic acid fragment encoding all or a portion of asoybean seed storage protein placed in sense or antisense orientationrelative to the promoter of (i), and (iii) a transcriptional terminationregion;

[0015] (b) creating a transgenic soybean cell by introducing into asoybean cell the chimeric gene of (a); and

[0016] (c) growing the transgenic soybean cells of step (b) underconditions that result in expression of the chimeric gene of step (a)

[0017] wherein the quantity of one or more members of a class of soybeanseed storage protein subunits is reduced when compared to soybeans notcontaining the chimeric gene of step (a).

DETAILED DESCRIPTION OF THE INVENTION BRIEF DESCRIPTION OF THE SEQUENCEDESCRIPTIONS

[0018] The invention can be more fully understood from the followingdetailed description and the Sequence Descriptions which form a part ofthis application. The Sequence Descriptions contain the three lettercodes for amino acids as defined in 37 C.F.R. 1.822 which areincorporated herein by reference.

[0019] SEQ ID NO:1 shows the 5′ to 3′ nucleotide sequence encoding the αsubunit of the β-conglycinin soybean seed storage protein.

[0020] SEQ ID NO:2 shows the 5′ to 3′ nucleotide sequence encoding theα′ subunit of the β-conglycinin soybean seed storage protein.

[0021] SEQ ID NO:3 shows the 5′ to 3′ nucleotide sequence encoding the βsubunit of the β-conglycinin soybean seed storage protein.

[0022] SEQ ID NOS:4 and 5 show the nucleotide sequences of the PCRprimers ConS and Con1.4a (respectively) used to isolate nucleic acidfragments encoding the α and α′ subunits of the β-conglycinin soybeanseed storage protein.

[0023] SEQ ID NOS:6 and 7 show nucleotide sequences of the PCR primersCon.09 and Con.8 (respectively) used to distinguish nucleic acidfragments encoding the α and α′ subunits of the β-conglycinin soybeanseed storage protein.

[0024] SEQ ID NOS:8 and 9 show the nucleotide sequences of the PCRprimers ConSa and Con1.9a (respectively) used to isolate full lengthcDNAs encoding the α and α′ subunits of the β-conglycinin soybean seedstorage protein.

[0025] SEQ ID NO:10 shows the nucleotide sequence of the PCR primerCon.1.0 used to confirm the full length cDNA encoding the α and α′subunits of the β-conglycinin soybean seed storage protein.

[0026] SEQ ID NOS:11, 12 and 13 show the 5′ to 3′ nucleotide sequencesencoding the Gy1, Gy2 and Gy3 subunits (respectively) of the group Iglycinin soybean seed storage protein.

[0027] SEQ ID NOS:14 and 15 show the 5′ to 3′ nucleotide sequencesencoding the Gy4 and Gy5 subunits (respectively) of the group IIglycinin soybean seed storage protein.

[0028] SEQ ID NOS:16, 17 and 18 show the nucleotide sequences of the PCRprimers G1-1, G1-1039 and G1-1475 (respectively) used to isolate thecDNAs encoding the subunits of the group I glycinin soybean seed storageprotein.

[0029] SEQ ID NOS:19, 20 and 21 show the nucleotide sequences of the PCRprimers G4-7, G4-1251, and G4-1670 (respectively) used to isolate thecDNA encoding the subunits of the group II glycinin soybean seed storageprotein.

BRIEF DESCRIPTION OF THE FIGURES

[0030]FIG. 1 is a restriction map of plasmid pML70, used as anintermediate cloning vehicle in construction of chimeric genes of theinstant invention.

[0031]FIG. 2 is a restriction map of plasmid pCW109, used as anintermediate cloning vehicle in construction of chimeric genes of theinstant invention.

[0032]FIG. 3 is a restriction map of plasmid pKS18HH, used as anintermediate cloning vehicle in construction of chimeric genes of theinstant invention.

[0033]FIG. 4 is a restriction map of plasmid pJo1. This plasmid wasderived by cloning the plant transcriptional unit KTi promoter/truncateda subunit of β-conglycinin/KTi 3′ end into the BamH I site of pKS18HH.

[0034]FIG. 5 is an SDS-PAGE gel of extracted protein from somaticembryos transformed with pJo1.

[0035]FIG. 6 is a restriction map of plasmid pBS43. This plasmidcomprises a nucleic acid sequence encoding the Glycine max microsomaldelta-12 desaturase under the transcriptional control of the soybeanα-conglycinin promoter.

[0036]FIG. 7 is an SDS-PAGE gel of extracted protein from soybean seedsobtained from plants transformed with pBS43.

[0037]FIG. 8 is a restriction map of plasmid pJo3. This plasmid wasderived by cloning the plant transcriptional unit KTi promoter/fulllength cDNA of the α subunit of β-conglycinin/KTi 3′ end into theHindIII site of pKS18HH.

[0038]FIG. 9 is a restriction map of plasmid pRB20. This plasmid wasderived by cloning the transcriptional unit β-conglycininpromoter/Phaseolin 3′ end into the HindIII site of pKS18HH. It is usedas an intermediate cloning vehicle in construction of chimeric genes ofthe instant invention.

Biological Deposits

[0039] The following plasmids have been deposited under the terms of theBudapest Treaty at American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209, and bear the followingaccession numbers: Plasmid Accession Number Date of Deposit pJo1 ATCC97614 Jun. 15, 1996 pBS43 ATCC 97619 Jun. 19, 1996 pJo3 ATCC 97615 Jun.15, 1996

Definitions

[0040] In the context of this disclosure, a number of terms shall beused. The term “nucleic acid” refers to a large molecule which can besingle-stranded or double-stranded, composed of monomers (nucleotides)containing a sugar, a phosphate and either a purine or pyrimidine. A“nucleic acid fragment” is a fraction of a given nucleic acid molecule.In higher plants, deoxyribonucleic acid (DNA) is the genetic materialwhile ribonucleic acid (RNA) is involved in the transfer of theinformation in DNA into proteins. A “genome” is the entire body ofgenetic material contained in each cell of an organism. The term“nucleotide sequence” refers to the sequence of DNA or RNA polymers,which can be single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases capable ofincorporation into DNA or RNA polymers.

[0041] As used herein, the term “homologous to” refers to therelatedness between the nucleotide sequence of two nucleic acidmolecules or between the amino acid sequences of two protein molecules.Estimates of such homology are provided by either DNA-DNA or DNA-RNAhybridization under conditions of stringency as is well understood bythose skilled in the art (Hames and Higgins, Eds. (1985) Nucleic AcidHybridisation, IRL Press, Oxford, U.K.); or by the comparison ofsequence similarity between two nucleic acids or proteins, such as bythe method of Needleman et al. ((1970) J. Mol. Biol. 48:443-453).

[0042] As used herein, “essentially similar” refers to DNA sequencesthat may involve base changes that do not cause a change in the encodedamino acid, or which involve base changes which may alter one or moreamino acids, but do not affect the functional properties of the proteinencoded by the DNA sequence. It is therefore understood that theinvention encompasses more than the specific exemplary sequences.Modifications to the sequence, such as deletions, insertions, orsubstitutions in the sequence which produce silent changes that do notsubstantially affect the functional properties of the resulting proteinmolecule are also contemplated. For example, alteration in the genesequence which reflect the degeneracy of the genetic code, or whichresults in the production of a chemically equivalent amino acid at agiven site, are contemplated; thus, a codon for the amino acid alanine,a hydrophobic amino acid, may be substituted by a codon encoding anotherhydrophobic amino acid residue such as glycine, valine, leucine, orisoleucine. Similarly, changes which result in substitution of onenegatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, can also be expected to produce a biologicallyequivalent product. Nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the protein molecule would alsonot be expected to alter the activity of the protein. In some cases, itmay in fact be desirable to make mutants of the sequence in order tostudy the effect of alteration on the biological activity of theprotein. Each of the proposed modifications is well within the routineskill in the art, as is determination of retention of biologicalactivity of the encoded products. Moreover, the skilled artisanrecognizes that “essentially similar” sequences encompassed by thisinvention can also defined by their ability to hybridize, understringent conditions (0.1×SSC, 0.1% SDS, 65° C.), with the sequencesexemplified herein.

[0043] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding) and following (3′ non-coding) the coding region. “Native”gene refers to an isolated gene with its own regulatory sequences asfound in nature. “Chimeric gene” refers to a gene that comprisesheterogeneous regulatory and coding sequences not found in nature.“Endogenous” gene refers to the native gene normally found in itsnatural location in the genome and is not isolated. A “foreign” generefers to a gene not normally found in the host organism but that isintroduced by gene transfer.

[0044] “Coding sequence” or “coding region” refers to a DNA sequencethat codes for a specific protein and excludes the non-coding sequences.It may constitute an “uninterrupted coding sequence”, i.e., lacking anintron or it may include one or more introns bounded by appropriatesplice junctions. An “intron” is a nucleotide sequence that istranscribed in the primary transcript but that is removed throughcleavage and re-ligation of the RNA within the cell to create the maturemRNA that can be translated into a protein.

[0045] “Initiation codon” and “termination codon” refer to a unit ofthree adjacent nucleotides in a coding sequence that specifiesinitiation and chain termination, respectively, of protein synthesis(mRNA translation). “Open reading frame” refers to the coding sequenceuninterrupted by introns between initiation and termination codons thatencodes an amino acid sequence.

[0046] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA. “Antisense RNA” refers to a RNAtranscript that is complementary to all or part of a target primarytranscript or mRNA and that blocks the expression of a target gene. Thecomplementarity of an antisense RNA may be with any part of the specificgene transcript, i.e., at the 5′ non-coding sequence, 3′ non-codingsequence, introns, or the coding sequence.

[0047] As used herein, “suitable regulatory sequences” refer tonucleotide sequences in native or chimeric genes that are locatedupstream (5′), within, or downstream (3′) to the nucleic acid fragmentsof the invention, which control the expression of the nucleic acidfragments of the invention. The term “expression”, as used herein,refers to the transcription and stable accumulation of the sense (mRNA)or the antisense RNA derived from the nucleic acid fragment(s) of theinvention that, in conjunction with the protein apparatus of the cell,results in altered phenotypic traits. Expression of the gene involvestranscription of the gene and translation of the mRNA into precursor ormature proteins. “Antisense inhibition” refers to the production ofantisense RNA transcripts capable of preventing the expression of thetarget protein. “Overexpression” refers to the production of a geneproduct in transgenic organisms that exceeds levels of production innormal or non-transformed organisms. “Cosuppression” refers to theexpression of a foreign gene which has substantial homology to anendogenous gene resulting in the suppression of expression of both theforeign and the endogenous gene. “Altered levels” refers to theproduction of gene product(s) in transgenic organisms in amounts orproportions that differ from that of normal or non-transformedorganisms. The skilled artisan will recognize that the phenotypiceffects contemplated by this invention can be achieved by alteration ofthe level of gene product(s) produced in transgenic organisms relativeto normal or non-transformed organisms, namely a reduction in geneexpression mediated by antisense suppression or cosuppression.

[0048] “Promoter” refers to a DNA sequence in a gene, usually upstream(5′) to its coding sequence, which controls the expression of the codingsequence by providing the recognition for RNA polymerase and otherfactors required for proper transcription. In artificial DNA constructs,promoters can also be used to transcribe antisense RNA. Promoters mayalso contain DNA sequences that are involved in the binding of proteinfactors which control the effectiveness of transcription initiation inresponse to physiological or developmental conditions. It may alsocontain enhancer elements. An “enhancer” is a DNA sequence which canstimulate promoter activity. It may be an innate element of the promoteror a heterologous element inserted to enhance the level ortissue-specificity of a promoter. “Constitutive promoters” refers tothose that direct gene expression in all tissues and at all times.“Tissue-specific” or “development-specific” promoters as referred toherein are those that direct gene expression almost exclusively inspecific tissues, such as leaves or seeds, or at specific developmentstages in a tissue, such as in early or late embryogenesis,respectively.

[0049] The “3′ non-coding sequences” refers to the DNA sequence portionof a gene that contains a polyadenylation signal and any otherregulatory signal capable of affecting mRNA processing or geneexpression. The polyadenylation signal is usually characterized byaffecting the addition of polyadenylic acid tracts to the 3′ end of themRNA precursor.

[0050] The term “operably linked” refers to nucleic acid sequences on asingle nucleic acid molecule which are associated so that the functionof one is affected by the other. For example, a promoter is operablylinked with a structural gene when it is capable of affecting theexpression of that structural gene (i.e., that the structural gene isunder the transcriptional control of the promoter).

[0051] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritence. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” organisms.

[0052] This invention concerns the construction of transgenic soybeanlines wherein the expression of genes encoding seed storage proteins aremodulated to effect a change in seed storage protein profile oftransgenic plants. Modification of the seed storage protein profile canresult in production of novel soy protein products with unique andvaluable functional characteristics.

[0053] Gene expression in plants uses regulatory sequences that arefunctional in such plants. The expression of foreign genes in plants iswell-established (De Blaere et al. (1987) Meth. Enzymol. 153:277-291).The source of the promoter chosen to drive the expression of thefragments of the invention is not critical provided it has sufficienttranscriptional activity to accomplish the invention by decreasing theexpression of the target seed storage protein genes. Preferred promotersinclude strong constitutive plant promoters, such as those directing the19S and 35S transcripts in cauliflower mosaic virus (Odell, J. T. et al.(1985) Nature 313:810-812; Hull et al. (1987) Virology 86:482-493).Particularly preferred promoters are those that allow seed-specificexpression. Examples of seed-specific promoters include, but are notlimited to, the promoters of seed storage proteins, which can representup to 90% of total seed protein in many plants. The seed storageproteins are strictly regulated, being expressed almost exclusively inseeds in a highly tissue-specific and stage-specific manner (Higgins etal. (1984) Ann. Rev. Plant Physiol. 35:191-221; Goldberg et al. (1989)Cell 56:149-160). Moreover, different seed storage proteins may beexpressed at different stages of seed development.

[0054] Expression of seed-specific genes has been studied in greatdetail (See reviews by Goldberg et al. (1989) Cell 56:149-160 andHiggins et al. (1984) Ann. Rev. Plant Physiol. 35:191-221). There arecurrently numerous examples of seed-specific expression of seed storageprotein genes (natural or chimeric) in transgenic dicotyledonous plants;in general, temporal and spatial expression patterns are maintained. Thepromoters used in such examples could potentially be used to affect thepresent invention. These include genes from dicotyledonous plants forbean β-phaseolin (Sengupta-Gopalan et al.(1985) Proc. Natl. Acad. Sci.USA 82:3320-3324; Hoffman et al. (1988) Plant Mol. Biol. 11:717-729),bean lectin (Voelker et al. (1987) EMBO J. 6:3571-3577), soybean lectin(Okamuro et al. (1986) Proc. Natl. Acad. Sci. USA 83:8240-8244), soybeanKunitz trypsin inhibitor (Perez-Grau et al. (1989) Plant Cell1:095-1109), soybean β-conglycinin (Beachy et al. (1985) EMBO J.4:3047-3053; pea vicilin (Higgins et al. (1988) Plant Mol. Biol.11:683-695), pea convicilin (Newbigin et al. (1990) Planta 180:461-470),pea legumin (Shirsat et al. (1989) Mol. Gen. Genetics 215:326-331),rapeseed napin (Radke et al. (1988) Theor. Appl. Genet. 75:685-694) andArabidopsis thaliana 2S albumin (Vandekerckhove et al. (1989)Bio/Technology 7:929-932).

[0055] Of particular use in the expression of the nucleic acid fragmentof the invention will be the heterologous promoters from several soybeanseed storage protein genes such as those for the Kunitz trypsininhibitor (KTi; Jofuku et al. (1989) Plant Cell 1:1079-1093; glycinin(Nielson et al. (1989) Plant Cell 1:313-328), and β-conglycinin (Haradaet al. (1989) Plant Cell 1:415-425). The skilled artisan will recognizethat attention must be paid to differences in temporal regulationendowed by different seed promoters. For example, the promoter for theα-subunit gene is expressed a few days before that for the β-subunitgene (Beachy et al. (1985) EMBO J. 4:3047-3053), so that the use of theβ-subunit gene is likely to be less useful for suppressing α-subunitexpression.

[0056] Also of potential use, but less preferred, will be the promotersof genes involved in other aspects of seed metabolism, such as lipid orcarbohydrate biosynthesis. In summary, the skilled artisan will have nodifficulty in recognizing that any promoter of sufficient strength andappropriate temporal expression pattern can potentially be used toimplement the present invention. Similarly, the introduction ofenhancers or enhancer-like elements into the promoter regions of eitherthe native or chimeric nucleic acid fragments of the invention wouldresult in increased expression to accomplish the invention. This wouldinclude viral enhancers such as that found in the 35S promoter (Odell etal. (1988) Plant Mol. Biol. 10:263-272), enhancers from the opine genes(Fromm et al. (1989) Plant Cell 1:977-984), or enhancers from any othersource that result in increased transcription when placed into apromoter operably linked to the nucleic acid fragment of the invention.

[0057] Of particular importance is the DNA sequence element isolatedfrom the gene encoding the α-subunit of β-conglycinin that can confer a40-fold, seed-specific enhancement to a constitutive promoter (Chen etal. (1989) Dev. Genet. 10:112-122). One skilled in the art can readilyisolate this element and insert it within the promoter region of anygene in order to obtain seed-specific enhanced expression with thepromoter in transgenic plants. Insertion of such an element in anyseed-specific gene that is normally expressed at times different thanthe β-conglycinin gene will result in expression of that gene intransgenic plants for a longer period during seed development.

[0058] Any 3′ non-coding region capable of providing a polyadenylationsignal and other regulatory sequences that may be required for theproper expression of the nucleic acid fragments of the invention can beused to accomplish the invention. This would include 3′ ends of thenative fatty acid desaturase(s), viral genes such as from the 35S or the19S cauliflower mosaic virus transcripts, from the opine synthesisgenes, ribulose 1,5-bisphosphate carboxylase, or chlorophyll a/b bindingprotein. There are numerous examples in the art that teach theusefulness of different 3′ non-coding regions.

[0059] Various methods of transforming cells of higher plants accordingto the present invention are available to those skilled in the art (seeEuropean Patent Publications EP-A-295,959 and EP-A-318,341). Suchmethods include those based on transformation vectors utilizing the Tiand Ri plasmids of Agrobacterium spp. It is particularly preferred touse the binary type of these vectors. Ti-derived vectors transform awide variety of higher plants, including monocotyledonous anddicotyledonous plants (Sukhapinda et al. (1987) Plant Mol. Biol.8:209-216; Potrykus, (1985) Mol. Gen. Genet. 199:183). Othertransformation methods are available to those skilled in the art, suchas direct uptake of foreign DNA constructs (see European PatentPublication EP-A-295,959), techniques of electroporation (Fromm et al.(1986) Nature (London) 319:791) or high-velocity ballistic bombardmentwith metal particles coated with the nucleic acid constructs (Klein etal. (1987) Nature (London) 327:70). Once transformed, the cells can beregenerated by those skilled in the art. Of particular relevance are therecently described methods to transform soybean, including McCabe et al.((1988) Bio/Technology 6:923-926), Finer et al. ((1991) In Vitro Cell.Dev. Biol. 27:175-182) and Hinchee, M. A. W. ((1988) Bio/Technology6:915-922).

[0060] Once transgenic plants are obtained by one of the methodsdescribed above, it is necessary to screen individual transgenics forthose that most effectively display the desired phenotype. It is wellknown to those skilled in the art that individual transgenic plantscarrying the same construct may differ in expression levels; thisphenomenon is commonly referred to as “position effect”. Thus, in thepresent invention different individual transformants may vary in theeffectiveness of suppression of the target seed protein. The personskilled in the art will know that special considerations are associatedwith the use of antisense or cosuppresion technologies in order toreduce expression of particular genes. U.S. Pat. Nos. 5,190,931,5,107,065 and 5,283,323 have taught the feasibility of these techniques,but it is well known that their efficiency is unpredictable.Accordingly, the person skilled in the art will make multiple geneticconstructs containing one or more different parts of the gene to besuppressed, since the art does not teach a method to predict which willbe most effective for a particular gene. Furthermore, even the mosteffective constructs will give an effective suppression phenotype onlyin a fraction of the individual transgenic lines isolated. For example,World Patent Publications WO93/11245 and WO94/11516 teach that whenattempting to suppress the expression of fatty acid desaturase genes incanola, actual suppression was obtained in less than 1% of the linestested. In other species the percentage is somewhat higher, but in nocase does the percentage reach 100. This should not be seen as alimitation on the present invention, but instead as practical matterthat is appreciated and anticipated by the person skilled in this art.Accordingly, the skilled artisan will develop methods for screeninglarge numbers of transformants. The nature of these screens willgenerally be chosen on practical grounds, and is not an inherent part ofthe invention. A preferred method will be one which allows large numbersof samples to be processed rapidly, since it will be expected that themajority of samples will be negative.

[0061] The mechanism of cosuppression remains unclear (for one reviewand speculation, see Flavell, R. (1994) Proc. Natl. Acad. Sci. USA91:3490-3496), and therefore the exact requirments to induce it whendesired are also unclear. Most examples found in the literature involvethe use of all or a large part of the transcribed region of the gene tobe cosuppressed to elicit the desired response. However, in at least onecase (Brusslan et al. (1993) Plant Cell 5:667-677; Brusslan and Tobin(1995) Plant Mol. Biol. 27:809-813), that of the cab140 gene ofArabidopsis, the use of the promoter (as a 1.3 kb fragment) and just 14bp of transcribed region fused to a completely unrelated gene wassufficient to result in cosuppression of the endogenous cabl40 gene aswell as the introduced chimeric gene. This result is unusual andapparantly quite unpredictable, as numerous other promoter-leader (the5′ untranslated leader being defined as the region between the start oftranscription and the translation initiation codon) units have been usedto drive chimeric genes successfully. Flavell speculates that some ormany genes (including members of multigene families such as thoseencoding seed proteins) may have evolved so as to avoid the mechanismsof cosuppression, while others have not, providing a potential furtherlevel of regulation as genomes evolve. Thus, the instant observationthat the promoter and leader of the conglycinin gene can be used tosuppress expression of endogenous conglycins while the other portion ofthe transgene (beyond the initiation codon) can be used to suppress acompletely unrelated gene is unique.

EXAMPLES

[0062] The present invention is further defined by the followingexamples. It will be understood that the examples are given forillustration only and the present invention is not limited to usesdescribed in the examples. The present invention can be used to generatetransgenic soybean plants with altered levels of various seed storageproteins. From the above discussion and the following examples, oneskilled in the art can ascertain, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions. All suchmodifications are intended to fall within the scope of the intendedclaims.

[0063] Detailed procedures for DNA manipulation, such as use ofrestriction endonuclease enzymes, other modifying enzymes, agarose gelelectrophoresis, nucleic acid hybridization, and transformation of E.coli with plasmid DNA are described in Sambrook et al. (1989) MolecularCloning, A Laboratory manual, 2nd ed, Cold Spring Harbor LaboratoryPress (hereinafter “Maniatis”). All restriction enzymes and othermodifying enzymes were obtained from Gibco BRL (Gaithersburg, Md.).

Example 1

[0064] To determine whether the expression of β-conglycinin indeveloping soybean cotyledons could be the target of cosuppression,truncated cDNA fragments of the α and α′ subunits of β-conglycinin wereprepared using a reverse transcriptase polymerase chain reaction kit(Geneamp™ RNA PCR Kit; Perkin Elmer Cetus). The upper primer, ConS, ishomologous to nucleotides 5-19 of the α and α′ subunit cDNA sequencesobtained from the EMBL/GenBank/DDBJ databases. To aid cloning,additional nucleotides were added to the 5′ end to code for an Nco Irestriction site. The lower primer, Con 1.4a, is complementary tonucleotides 1370-1354 of SEQ ID NO:1 and 1472-1456 of SEQ ID NO:2,representing the sequences of the α and α′ cDNAs, respectively. To aidin cloning, additional nucleotides were added to the 5′ end to introducea Kpn I restriction site. The nucleotide sequences of PCR primers ConSand Con1.4a are shown below. ConS 5′-CGTACCATGGTGAGAGCGCGGTTCC-3′ (SEQID NO:4)         Nco I Con1.4a 5′-CGGTACCGAATTGAAGTGTGGTAG-3′ (SEQ IDNO:5)       Kpn I

[0065] RNA isolated from developing soybean seeds wasreverse-transcribed using either the kit-supplied random hexamers, orCon1.4a, following the manufacturer's protocol. The resulting cDNAfragments were amplified in the PCR (Polymerase Chain Reaction) reactionusing a mixture of ConS and Con1.4a. Reactant concentrations were asdescribed in the manufacturer's protocols. The following program wasused: a) one cycle of 2 minutes at 95° C.; b) 35 cycles of: 1.5 minutesat 50° C. (annealing), 5 minutes at 70° C. (extension), 1.5 minutes at95° C. (denaturation); and c) one cycle of 2 minutes at 50° C. followedby 10 minutes at 68° C. Fifteen microliters of each of the PCR reactionmixes was analyzed by agarose gel electrophoresis. Reactions resulted inPCR products of the expected sizes: 1.47 kb for α′ and 1.37 kb for α.The truncated cDNA fragments from the remainder of the reaction mixeswere purified using the Wizard™ PCR Preps DNA Purification System kit(Promega).

[0066] The purified reaction mix containing the α and α′ fragments,which because of the primers used, included Nco I restriction sites atthe 5′ ends and Kpn I restriction sites at the 3′ ends, were digestedwith Kpn I and Nco I restriction enzymes. The a cDNA fragment wasrecovered following gel electrophoresis, designated as fragment F8, anddirectionally cloned (sense orientation) into pCW109 (FIG. 1) and pML70(FIG. 2) using the Nco I to Kpn I sites present in both plasmids. F8 wasconfirmed as a by PCR using a nested set of primers (Con.09 and Con.8)internal to ConS and Con1.4a, and distinguished from α′ by digestion ofpCW109/F8 plasmid with Hind III, Nco I, Kpn I, and Pst I (α does notcontain a Pst I site whereas α′ does). Con.095′-TCGTCCATGGAGCGCGGTTCCCATTAC-3′ (SEQ ID NO:6) Con.85′-TCTCGGTCGTCGTTGTT-3′ (SEQ ID NO:7)

[0067] The transcriptional unit KTi promoter/truncated α/KTi 3′ end wasreleased from plasmid pML70/F8 by restriction digest with BamHI, gelisolated, and labeled as F11. F11 was then cloned into pKS18HH (FIG. 3)at the BamH I site. pKS18HH is a plasmid construction containing thefollowing genetic elements: (i) T7 promoter/Hygromycin BPhosphotransferase (HPT)/T7 Terminator Sequence; (ii) 35S promoter fromcauliflower mosaic virus (CaMV)/Hygromycin B Phosphotransferase(HPT)/Nopaline Synthase (NOS) 3′ from Agrobacterium tumefaciens T-DNA;and (iii) pSP72 plasmid vector (Promega) with beta-lactamase codingregion removed. One skilled in the art of molecular biology can ligatethe above three components into a single plasmid vector using well knownprotocols (Maniatis).

[0068] The Hygromycin B Phosphotransferase (HPT) gene was isolated byPCR amplification from E. coli strain W677 containing aKlebsiella-derived plasmid pJR225 (Gritz L., and Davies J. (1983) Gene25:179-188). pKS18HH contains the CaMV 35S/HPT/NOS cassette forconstitutive expression of the HPT enzyme in plants, such as soybean.The pKS18HH plasmid also contains the T7 promoter/HPT/T7 terminatorcassette for expression of the HPT enzyme in certain strains of E. coli,such as NovaBlue™ (DE3) (Novagen) that are lysogenic for lambda DE3(which carries the T7 RNA Polymerase gene under lacUV5 control). pKS18HHalso contains three unique restriction endonuclease sites suitable forcloning of genes into this vector. Thus, the pKS18HH plasmid vectorallows the use of Hygromycin B for selection in both E. coli and plants.Confirmation of insertion and orientation of the F11 fragment wasaccomplished by digestion with HindIII. A clone with the F11 fragment inclockwise orientation was selected and labeled pJo1 (FIG. 4).

Transformation of Somatic Embryo Cultures

[0069] The following stock solutions and media were used fortransformation and propogation of soybean somatic embryos: StockSolutions (g/L) Media MS Sulfate 100x stock SB55 (per Liter) MgSO₄ 7H₂O37.0 10 mL of each MS stock MnSO₄ H₂O 1.69 1 mL of B5 Vitamin stockZnSO₄ 7H₂O 0.86 0.8 g NH₄NO₃ CuSO₄ 5H₂O 0.0025 3.033 g KNO₃ 1 mL 2,4-D(10 mg/mL stock) MS Halides 100x stock CaCl₂ 2H₂O 44.0 0.667 gasparagine KI 0.083 pH 5.7 CoCl₂ 6H₂O 0.00125 KH₂PO₄ 17.0 SB103 (perLiter) H₃BO₃ 0.62 1 pk. Murashige & Skoog salt mixture (Gibco BRL)Na₂MoO₄ 2H₂O 0.025 60 g maltose Na₂EDTA 3.724 2 g gelrite FeSO₄ 7H₂O2.784 pH 5.7 (For SB103 plus charcoal, add 5 g charcoal) B5 Vitaminstock SB148 (per Liter) myo-inositol 100.0 1 pk. Murashige & Skoog saltmixture (Gibco BRL) nicotinic acid 1.0 60 g maltose pyridoxine HCl 1.0 1mL B5 vitamin stock thiamine 10.0 7 g agarose pH 5.7

[0070] Soybean embryonic suspension cultures were maintained in 35 mLliquid media (SB55) on a rotary shaker (150 rpm) at 28° C. with a mix offluorescent and incandescent lights providing a 16/8 h day/nightschedule. Cultures were subcultured every 2 to 3 weeks by inoculatingapproximately 35 mg of tissue into 35 mL of liquid media.

[0071] Soybean embryonic suspension cultures were transformed with pJo1by the method of particle gun bombardment (see Klein et al. (1987)Nature 327:70). A DuPont Biolistic™ PDS1000/He instrument was used forthese transformations.

[0072] Five μL of pJo1 plasmid DNA (1 μg/μL), 50 μL CaCl₂ (2.5 M), and20 μL spermidine (0.1 M) were added to 50 μL of a 60 mg/mL 1 mm goldparticle suspension. The particle preparation was agitated for 3minutes, spun in a microfuge for 10 seconds and the supernatant removed.The DNA-coated particles were then washed once with 400 μL 70% ethanoland resuspended in 40 μL of anhydrous ethanol. The DNA/particlesuspension was sonicated three times for 1 second each. Five μL of theDNA-coated gold particles were then loaded on each macro carrier disk.

[0073] Approximately 300 to 400 mg of two week old suspension culturewas placed in an empty 60 mm×15 mm petri dish and the residual liquidremoved from the tissue by pipette. The tissue was placed about 3.5inches away from the retaining screen and bombarded twice. Membranerupture pressure was set at 1000 psi and the chamber was evacuated to−28 inches of Hg. Two plates were bombarded per construct perexperiment. Following bombardment, the tissue was divided in half andplaced back into liquid media and cultured as described above.

[0074] Fifteen days after bombardment, the liquid media was exchangedwith fresh SB55 containing 50 mg/mL hygromycin. The selective media wasrefreshed weekly. Six weeks after bombardment, green, transformed tissuewas isolated and inoculated into flasks to generate new transformedembryonic suspension cultures.

[0075] Transformed embryonic clusters were removed from liquid culturemedia and placed on a solid agar media, SB103, plus 0.5% charcoal tobegin maturation. After 1 week, embryos were transferred to SB103 mediaminus charcoal. After 3 weeks on SB103 media, maturing embryos wereseparated and placed onto SB148 media. Conditions during embryomaturation were 26° C., with a mix of fluorescnt and incandescent lightsproviding a 16/8 h day/night schedule. After 6 weeks on SB148 media,embryos were analyzed for the expression of the β-conglycinin subunitproteins. Each embryonic cluster gave rise to 5 to 20 somatic embryos.

Analysis of Transformed Somatic Embryos

[0076] Initial experiments were performed to determine when the α, α′and β subunits of β-conglycinin could be visualized during somaticembryo maturation by SDS-PAGE gel electrophoresis. Cotyledons ofnon-transformed embryos (generated as above, except they did not undergobombardment) were dissected from embryos at 6, 8, 10, and 12 weeks afterinitiating maturation and kept frozen at −80° C. until analyzed.Cotyledonary tissue was weighed, 10 μL/mg tissue of extraction bufferwas added, and the tissue ground in a Pellet Pestle Disposable Mixer(Kimble/Kontes). Extraction buffer consisted of 50 mM Tris-HCl (pH 7.5),10 mM β-mercaptoethanol (BME), and 0.1% SDS. The samples were thenmicrofuged at 12,000 rpm for 10 minutes and supernatant remove to a newmicrofuge tube by pipette. Extracts were kept frozen at −20° C. untilused.

[0077] For SDS-PAGE analysis, 8 μL of (2×) loading buffer was added to 8μL of sample extract. The (2×) loading buffer consisted of 100 mMTris-HCl (pH 7.5), 4% SDS, 0.2% bromophenol blue, 15% glycerol, and 200mM PME. The mixture was heated at 95° C. for 4 minutes. Sample mixeswere then microfuged (12,000 rpm for 20 seconds) and loaded onto a 10%precast Ready Gel™ (Bio-Rad) that was assembled into a mini-Protein IIElectrophoresis Cell (Bio-Rad). Bio-Rad Tris/Glycine/SDS Buffer was usedas the running buffer and voltage was a constant 125V. In addition tosample extracts, each gel contained one lane with a molecular weightstandard (Bio-rad SDS-PAGE standard, low range) and one lane with totalsoybean seed protein extracted from commercial defatted soy flour. Uponcompletion, the gels were stained with Coomassie Brilliant Blue anddestained (Maniatis) in order to visualize proteins. Gels werephotographed, placed in a sealed bag with water, and stored in therefrigerator. Results indicated that the α, α′ and β subunits ofβ-conglycinin were detectable in the cotyledons of somatic embryosbetween 8 and 10 weeks after the start of maturation.

[0078] Analysis of transformed embryos was carried out at 10 weeks afterthe start of maturation using the methods described above. Two embryosper clone were analyzed initially. Additional embryos were analyzed ifsuppression of the β-conglycinin subunits observed in the two embryos.Table 1 presents the results of this analysis, wherein the presence orabsence of each β-conglycinin subunit is indicated by a (+) or (−),respectively. TABLE 1 Clone Embryo α α′ β Jo1-1 1 − − + 2 − − + 3 + + +4 − − + 5 + + + Jo1-2 1 + + + 2 + + + Jo1-3 1 + + + 2 + + + Jo1-4 1 − −− 2 − − − 3 − − − 4 + + + 5 − − − Jo1-5b 1 + + + 2 + + + Jo1-5c 1 − − +2 − − + Jo1-5d 1 + + + 2 + + + Jo1-6a 1 − − + 2 − − + 3 − − + 4 − − +5 + + + Jo1-6b 1 + + + 2 + + + Jo1-6c 1 + + + Jo1-6d 1 + + + 2 + + +Jo1-6d 1 + + + 2 + + + Jo1-6e 1 + + + 2 + + + Jo1-7a 1 − − + 2 + + +Jo1-7b 1 − − + Jo1-8a 1 + + + Jo1-8b 1 + + + 2 + + + Jo1-9a 1 + + +2 + + + Jo1-9b 1 + + + 2 − − + Jo1-9c 1 + + + Jo1-10 1 − − + 2 + + +

[0079] Seven transgenic clones gave rise to embryos in which theexpression a and α′ was suppressed. In addition, one clone (Jo1-4) gaverise to embryos in which all three β-conglycinin subunits weresuppressed. This result is surprising as the truncated α transgenesequence overlaps with only a 0.75 kb portion of the total 1.32 kb βsubunit cDNA. Overall, there is only 52% similarity between thetruncated α transgene and the β subunit cDNA. With the knowledge athand, the truncated α transgene would not be considered to possesssufficient similarity of stucture to “cosuppress” the β subunit of theβ-conglycinin gene.

[0080] An example of an SDS-PAGE analysis is shown in FIG. 5. Lanes 1-3are extracts of three cotyledons dissected from embryos generated fromclone Jo1-1. Lanes 4 and 5 are protein molecular weight standards andsoy protein standard derived from seed, respectively. Lanes 6-8 areextracts of cotyledons dissected from embryos generated from cloneJo1-4. The protein pattern in lane 2 is an example of embryos in whichboth α and α′ are co-suppressed. The protein patterns in lanes 6 and 8are examples of embryos where all the subunits comprising β-conglycininare suppressed.

Example 2

[0081] To determine if expression of β-conglycinin could be suppressedin developing cotyledons by cosuppression using the β-conglycininpromoter region, a plasmid, designated pBS43, containing a Glycine maxmicrosomal delta-12 desaturase cDNA (GmFad 2-1) sequence (Heppard etal., (1996) Plant Physiol. 110:311-319; GenBank Acc. No. L43920) undercontrol of the soybean β-conglycinin promoter (Beachy et al., (1985)EMBO J. 4:3047-3053), was constructed. The construction of this vectorwas facilitated by the use of the following plasmids: pMH40, pCST2 andpBS13. The plasmid constructions detailed below are described in part inUnited States Patent Application No. U.S. Ser. No. 08/262,401 and WorldPatent Publication No. WO94/11516, both of which are incorporated hereinby reference.

[0082] The pMH40 vector was derived from plasmid pGEM9z, a commerciallyavailable cloning vector (Promega Biotech) by the insertion a 1.4 kb ³⁵Spromoter region from CaMV (Odell et al. (1985) Nature 303:810-812;Harpster et al. (1988) Mol. Gen. Genet. 212:182-190) coupled to theβ-glucuronidase gene from E. coli. This was a 1.85 kb fragment encodingthe enzyme β-glucuronidase (Jefferson et al. (1986) PNAS USA83:8447-8451) and a 0.3 kb DNA fragment containing the transcriptionterminator from the nopaline synthase gene of the Ti-plasmid ofAgrobacterium tumefaciens (Fraley et al. (1983) PNAS USA 80:4803-4807).

[0083] The vector pCST2 was derived from vectors pML18 and pCW109A. Theplasmid pCW109A contains the soybean β-conglycinin promoter sequence andthe phaseolin 3′ untranslated region and is a modified version of vectorpCW109 which was derived from the commercially available plasmid pUC18(Gibco-BRL). The vector pCW109 was made by inserting into the Hind IIIsite of the cloning vector pUC18a 555 bp 5′ non-coding region(containing the promoter region) of the β-conglycinin gene followed bythe multiple cloning sequence containing the restriction endonucleasesites for Nco I, Sma I, Kpn I and Xba I, then 1174 bp of the common beanphaseolin 3′ untranslated region into the Hind III site. Theβ-conglycinin promoter region used is an allele of the publishedβ-conglycinin gene (Doyle et al., (1986) J. Biol. Chem. 261:9228-9238)due to differences at 27 nucleotide positions. Further sequencedescription of this gene may be found World Patent PublicationWO91/13993.

[0084] To facilitate use in antisense constructions, the Nco I site andpotential translation start site in the plasmid pCW109 was destroyed bydigestion with Nco I, mung bean exonuclease digestion and religation ofthe blunt site to give the modified plasmid pCW109A.

[0085] The vector pML18 consists of the non-tissue specific andconstitutive cauliflower mosaic virus (35S) promoter (Odell et al.,(1985) Nature 313:810-812; Hull et al., (1987) Virology 86:482-493),driving expression of the neomycin phosphotransferase gene (Beck et al.(1982) Gene 19:327-336) followed by the 3′ end of the nopaline synthasegene including nucleotides 848 to 1550 (Depicker et al. (1982) J. Appl.Genet. 1:561-574). This transcriptional unit was inserted into thecommercial cloning vector pGEM9z (Gibco-BRL) and is flanked at the 5′end of the 35S promoter by the restriction sites Sal I, Xba I, Bam HIand Sma I, in that order. An additional Sal I site is present at the 3′end of the NOS 3′ sequence and the Xba I, Bam HI and Sal I sites areunique. The plasmid pML18 was digested with Xba I, the singled strandedends were filled-in using the Klenow fragment of DNA polymerase I, andthe product was ligated in order to remove the Xba I site. The resultingplasmid was designated pBS16.

[0086] The plasmid pCW109A was digested with Hind III and the resulting1.84 kb fragment, which contained the P-conglycinin/antisense delta-12desaturase cDNA/phaseolin 3′ untranslated region, was gel isolated. This1.84 kb fragment was ligated into the Hind III site of pBS16. A plasmidcontaining the insert in the desired orientation yielded a 3.53 kb and4.41 kb fragment when digested with Kpn I and this plasmid wasdesignated pCST2.

[0087] The vector pBS13 was used as the source of the GmFad2-1 cDNA,which encodes the soybean microsomal delta12-desaturase and possessesthe sequence as disclosed in GenBank Acc. No. L43920. The vector pBS13was derived from the vector pML70 (FIG. 1), which contains the KTi3promoter and the KTi3 3′ untranslated region and was derived from thecommercially available vector pTZ18R (Pharmacia) via the intermediateplasmids pML51, pML55, pML64 and pML65. A 2.4 kb Bst BI/Eco RI fragmentof the complete soybean KTi3 gene (Jofuku and Goldberg (1989) Plant Cell1:1079-1093), which contains all 2039 nucleotides of the 5′ untranslatedregion and 390 bases of the coding sequence of the KTi3 gene ending atthe Eco RI site corresponding to bases 755 to 761 of the sequencedescribed in Jofuku (supra), was ligated into the Acc I/Eco RI sites ofpTZ18R to create the plasmid pML51. To destroy an Nco I site in themiddle of the 5′ untranslated region of the KTi3 insert, plasmid pML51was cut with Nco I, the singled stranded ends were filled-in using theKlenow fragment of DNA polymerase I, and the product was religatedresulting in the plasmid pML55. The plasmid pML55 was partially digestedwith Xmn I/Eco RI to release a 0.42 kb fragment, corresponding to bases732 to 755 of the above cited sequence, which was discarded. A syntheticXmn I/Eco RI linker containing an Nco I site, was constructed by makinga dimer of complementary synthetic oligonucleotides consisting of thecoding sequence for an Xmn I site (5′-TCTTCC-3′) and an Nco I site(5′-CCATGGG-3′) followed directly by part of an Eco RI site(5′-GAAGG-3′). The Xmn I and Nco I/Eco RI sites were linked by a shortintervening sequence (5′-ATAGCCCCCCAA-3′). This synthetic linker wasligated into the Xmn I/Eco RI sites of the 4.94 kb fragment to createthe plasmid pML64. The 3′ untranslated region of the KTi3 gene wasamplified from the sequence described in Jofuku (supra) by standard PCRprotocols (Perkin Elmer Cetus, GeneAmp PCR kit) using the primers ML51and ML52. Primer ML51 contained the 20 nucleotides corresponding tobases 1072 to 1091 of the above cited sequence with the addition ofnucleotides corresponding to Eco RV (5-′GATATC-3′), Nco I(5′-CCATGG-3′), Xba I (5′-TCTAGA-3′), Sma I (5′-CCCGGG-3′) and Kpn I(5′-GGTACC-3′) sites at the 5′ end of the primer. Primer ML52 containedto the exact compliment of the nucleotides corresponding to bases 1242to 1259 of the above cited sequence with the addition of nucleotidescorresponding to Sma I (5′-CCCGGG-3′), Eco RI (5′-GAATTC-3′), Bam HI(5′-GGATCC-3′) and Sal I (5′-GTCGAC-3′) sites at the 5′ end of theprimer. The PCR-amplified 3′ end of the KTi3 gene was ligated into theNco I/Eco RI sites of pML64 to create the plasmid pML65. A syntheticmultiple cloning site linker was constructed by making a dimer ofcomplementary synthetic oligonucleotides consisting of the codingsequence for Pst I (5′-CTGCA-3′), Sal I (5′-GTCGAC-3′), Bam HI(5′-GGATCC-3′) and Pst I (5′-CTGCA-3′) sites. The linker was ligatedinto the Pst I site (directly 5′ to the KTi3 promoter region) of pML65to create the plasmid pML70.

[0088] The 1.46 kb Sma I/Kpn I fragment from soybean delta-12 desaturasecDNA, GmFad2-1 (GenBank Acc. No. L43920), was ligated into thecorresponding sites in pML70 resulting in the plasmid pBS10. Thedesaturase cDNA fragment was in the reverse (antisense) orientation withrespect to the KTi3 promoter in pBS10. The plasmid pBS10 was digestedwith Bam HI and a 3.47 kb fragment, representing the KTi3promoter/antisense desaturase cDNA/KTi3 3′ end transcriptional unit wasisolated by agarose gel electrophoresis. The vector pML18 consists ofthe non-tissue specific and constitutive cauliflower mosaic virus (35S)promoter (Odell et al., (1985) Nature 313:810-812; Hull et al., (1987)Virology 86:482-493), driving expression of the neomycinphosphotransferase gene (Beck et al. (1982) Gene 19:327-336) followed bythe 3′ end of the nopaline synthase gene including nucleotides 848 to1550 (Depicker et al. (1982) J. Appl. Genet. 1:561-574). Thistranscriptional unit was inserted into the commercial cloning vectorpGEM9z (Gibco-BRL) and is flanked at the 5′ end of the 35S promoter bythe restriction sites Sal I, Xba I, Bam HI and Sma I in that order. Anadditional Sal I site is present at the 3′ end of the NOS 3′ sequenceand the Xba I, Bam HI and Sal I sites are unique. The 3.47 kbtranscriptional unit released from pBS10 was ligated into the Bam HIsite of the vector pML18. When the resulting plasmids were digested withSma I and Kpn I, plasmids containing inserts in the desired orientationyielded 3 fragments of 5.74, 2.69 and 1.46 kb. A plasmid with thetranscriptional unit in the correct orientation was selected and wasdesignated pBS13.

[0089] The 1.46 kb XbaI/EcoRV fragment from pBS13 (described above) wasdirectionally cloned into the SmaI/XbaI site of vector pCST2 (describedabove) to yield a plasmid designated pBS39. The 3.3 kb HindIII fragmentof plasmid pBS39 was cloned into the HindIII site of plasmid pMH40(described above) to give the plant expression vector pBS43 (FIG. 6).

Transformation of Soybeans with Vector pBS43 and Identification of aTransgenic “Transwitch” Line

[0090] The vector pBS43 was transformed into soybean meristems using themethod of particle bombardment of soybean meristems (Christou et al(1990) Trends Biotechnol. 8:145-151). Seeds of transformed plants (i.e.,from plants which had been identified as positive for GUS activity) werescreened for fatty acid composition. Fatty acid methyl esters wereprepared from hexane extracts of small (approx. 10 mg) seed chips(Browse et al (1986) Anal. Biochem. 152:141-145). Seed chips from tendifferent transgenic lines were analysed and some of the R1 seeds fromone of these lines, designated 260-05, had a total oleic acid content of80-85% compared with about 20% in control seeds. This phenotype iscaused by the cosuppression of the endogenous Fad 2-1 gene and is theresult of the insertion of two copies of pBS43 into a locus of thesoybean genome designated the “Transwitch locus” (Kinney, A. J. (1995)in “Induced Mutations and Molecular Techniques for Crop Improvement”,International Atomic Energy Agency, Vienna). High oleic acid R1 seedsfrom line 260-05, which contained the Transwitch locus, were selfed andR2 seeds which were homozygous for the Transwitch locus were selected.Two of these R2 homozygous seeds (G94-1, G94-19) and seeds derived fromfurther generations of G94-1 and G94-19 (R3, R4, R5), were selected forfurther analysis.

[0091] R5 seeds of G94-1 and G94-19 plants grown in both Iowa and PuertoRico were ground into a powder and approximately 1 g extracted with 5 mLof hexane. After centifugation, the hexane was poured off and the flakesallowed to air dry. Approximately 10 mg of defatted powder was extractedas described above and analyzed by SDS-PAGE. In both transgenic linesderived from both locations, the expression of the α′ and α subunits ofβ-conglycinin were suppressed relative to control soybean lines and astandard soy flour (FIG. 7).

Example 3

[0092] To test if β-conglycinin expression could be suppressed usingantisense technology, full length cDNAs of α and α′ were made usingreverse transcriptase polymerase chain reaction as described above. Theupper primer, ConSa, is homologous to region 4-19 of both α and α′ cDNAsequences with additional nucleotides added to the 5′ end to code for aKpn I restriction site. The lower primer used, Con1.9a, is homologous toregions 1818-1801 of SEQ ID NO:1, representing the α isoform, and1920-1903 of SEQ ID NO:2, representing the α′ isoform, respectively. Toaid in subsequent cloning steps, additional nucleotides were added tothe 5′ end to code for an Nco I restriction site. ConSa5′-ACGGTACCGATGAGAGCGCGGTTCC-3′ (SEQ ID NO:8)       Kpn I Con1.9a5′-AACCCATGGTCAGTAAAAAGCCCTCAA-3′ (SEQ ID NO:9)        Nco I

[0093] Reverse transcription and subsequent PCR reaction were carriedout as described above. RNA isolated from developing soybean seeds wasreverse-transcribed using either random hexamers or Con1.9a (method asdetailed above). The cDNA was amplified in a PCR reaction using ConSaand Con1.9a using the protocol detailed above. Fifteen microliters ofthe PCR reaction mixes were analyzed by agarose gel electrophoresis. A1.8 kb band, the expected molecular weight for α, was observed. Theremaining reaction mixes were purified using Wizard™ PCR Preps DNAPurification System kit (Promega). The a cDNA, which because of theprimers used included a Kpn I site on the 5′ end and an Nco I site onthe 3′ end, was digested with Nco I and Kpn I restriction enzymes. Theresulting α cDNA was gel isolated, labeled as F10, and directionallycloned (antisense orientation) into pCW109 using the Nco I and Kpn Isites present in the plasmid. F10 was confirmed as a by PCR using nestedprimers (upper: Con.09 (SEQ ID NO:6); lower: Con1.4a (SEQ ID NO:5) andCon1.0 (SEQ ID NO:10)). Con1.0 5′-CGGGTATGGCGAGTGTT-3′ (SEQ ID NO:10)

[0094] The transcriptional unit β-conglycinin promoter/α cDNAantisense/phaseolin 3′ end was released from pCW109/F10 by partialdigest with Hind III. Conditions of the partial digest were such that 6fragments were produced (5.1 kb, 3.8 kb, 3.6 kb, 2.6 kb, 2.4 kb, and 1.2kb). The 3.6 kb fragment containing the the transcriptional unit was gelisolated and labeled F14. F14 was then cloned into the Hind III site ofpKS18HH. After confirming insertion by digestion of plasmid DNApreparations made from tansformed cells with Hind III, the plasmid DNAfrom positive cultures was digested with Kpn I to ensure that theycontained the 3.6 kb F14 fragment and not the 3.8 kb fragment from thepartial digest of pCW109/F10 with Hind III. F14 contains a Kpn I site,while the 3.8 kb fragment does not. Upon confirmation, pKS18HH/F14 waslabeled pJo3 (FIG. 8). Soybean embryonic suspension cultures weretransformed with pJo3 as detailed above. Transformation resulted in 5transformed clones; upon maturation each clone gave rise to 4 to 8somatic embryos.

[0095] Protein extracts of transformed somatic embryos were analyzed bySDS-PAGE as previously detailed. Results are presented in Table 2. Thetransgenic clones all gave rise to at least one somatic embryo in whichthe expression of both α and α′ was suppressed. TABLE 2 Clone Embryo αα′ β Jo3-1 1 − − + 2 + + + Jo3-2 1 − − + 2 − − + Jo3-2b 1 − − + 2 − − +Jo3-3 1 − − + 2 − − + Jo3-4 1 − − + 2 − − +

Example 4

[0096] There are five non allelic genes that code for the glycininsubunits.

[0097] Sequencing genomic clones and cDNA's have lead to a division ofthe subunit genes into two groups based on sequence similarity. Group Iconsists of Gy1 (SEQ ID NO:11), Gy2 (SEQ ID NO:12) and Gy3 (SEQ IDNO:13), whereas group II consists of Gy4 (SEQ ID NO:14) and Gy5 (SEQ IDNO:15). There is greater than 85% similarity between genes within agroup, but only 42% to 46% similarity between genes of different groups.To determine whether expression of glycinin can be suppressed indeveloping cotyledons by employing co-suppression technology, cDNA's ofGroup I and Group II were prepared using reverse transcriptasepolymerase chain reaction as described above.

[0098] The upper primer used for Group I reactions (G1-1) is homologusto regions 1-19 for all Group I cDNA's. Two lower primers were used:G1-1039, which is homologous with regions 1038-1022 of Gy1, 1008-992 ofGy2, and 996-980 of Gy3; or G1-1475, which is homologus to regions1475-1460 of Gy1, 1445-1430 of Gy2 and 1433-1418 of Gy3. To aid infuture cloning, all primers contained additional nucleotides that codedfor a Not I restriction site at their 5′ end. G1-15′-GCGGCCGCATGGCCAAGCTAGTTTTTT-3′ (SEQ ID NO:16)       Not I G1-10395′-GCGGCCGCTGGTGGCGTTTGTGA-3′ (SEQ ID NO:17)       Not I G1-14755′-GCGGCCGCTCTTCTGAGACTCCT-3′ (SEQ ID NO:18)       Not I

[0099] RNA isolated from developing soybean seeds wasreverse-transcribed using either random hexamers, or G1-1475 or G1-1039as the lower primer in the reactions. cDNA fragments were amplifiedusing a mixture of G1-1 with either G1-1039 or G1-1475. Fifteenmicroliters of the PCR reaction mixes were analyzed by agarose gelelectrophoresis. PCR reactions resulted in products of the expectedmolecular wieght, approximately 1 kb and 1.4-1.5 kb for primer setsG1-1/G1-1039 and G1-1/G1-1475, respectively. cDNA fragments from theremainder of the reaction mixes were purified using the Wizard™ PCRPreps DNA Purification System kit (Promega). Purified cDNA's were thendigested with Not I and isolated by agarose gel purification.

[0100] The upper primer used for RT-PCR reactions of Group II (G4-7) ishomologus to regions 7-22 for both cDNA's of Group II. Two lower primerswere used: G4-1251 which is homologus with regions 1251-1234 of Gy4 and1153-1135 of Gy5; or G4-1670 which is homologus to regions 1668-1653 ofGy4. There is no similar region in Gy5. To aid in future cloning allprimers contained additional nucleotides that coded for a Not Irestriction site at their 5′ end. G4-7 5′-GCGGCCGCATGCCCTTCACTCTCT-3′(SEQ ID NO:19)    Not I G4-1251 5′-GCGGCCGCTGGGAGGGTGAGGCTGTT-3′ (SEQ IDNO:20)    Not I G4-1670 5′-GCGGCCGCTGAGCCTTGTTGAGAC-3′ (SEQ ID NO:21)   Not I

[0101] RNA isolated from developing soybean seeds wasreverse-transcribed using either random hexamers, or G4-1251 or G4-1670as the lower primer in the reactions. cDNA fragments were amplifiedusing a mixture of G4-7 with either G4-1251 or G4-1670. Fifteenmicroliters of the PCR reaction mixes were analyzed by agarose gelelectrophoresis. PCR reactions resulted in products of the expectedmolecular weight, approximately 1.25 kb and 1.7 kb for primer setsG4-7/G4-1251 and G4-7/G4-16.70, respectively. cDNA fragments from theremainder of the reaction mixes were purified using the Wizard™ PCRPreps DNA Purification System kit (Promega). Purified cDNA's were thendigested with Not I and isolated from gels.

[0102] The isolated group I cDNAs are cloned into pRB20 (FIG. 9) at theNot I site (sense oritentation). After partial restriction digest withNot I and isolation of the single cut pRB20/group I linear fragments,group II cDNA are added to create final transcriptional unitsP-conglycinin promoter/group I cDNA (sense orientation)/phaseolin 3′ endand β-conglycinin promoter/group II cDNA (sense orientation)/phaseolin3′ end. The resulting plasmids are then used to transform somaticembryonic suspension cultures using the method detailed above.

1. A method for reducing the quantity of a soybean seed storage protein in soybean seeds comprising: (a) constructing a chimeric gene comprising: (i) a nucleic acid fragment encoding a promoter that is functional in the cells of soybean seeds; (ii) a nucleic acid fragment encoding all or a portion of a soybean seed storage protein placed in sense or antisense orientation relative to the promoter of (i); and (iii) a transcriptional termination region; (b) creating a transgenic soybean cell by introducing into a soybean cell the chimeric gene of (a); and (c) growing the transgenic soybean cells of step (b) under conditions that result in expression of the chimeric gene of step (a) wherein the quantity of one or more members of a class of soybean seed storage protein subunits is reduced when compared to soybeans not containing the chimeric gene of step (a).
 2. The method of claim 1 wherein the soybean seed storage protein is selected from the group consisting of glycinin and β-conglycinin.
 3. The method of claim 1 wherein the nucleic acid fragment encoding all or a portion of a soybean seed storage protein is placed in sense orientation relative to the promoter region.
 4. The method of claim 1 wherein the nucleic acid fragment encoding all or a portion of a soybean seed storage protein is placed in antisense orientation relative to the promoter region.
 5. The method of claim 4 wherein the nucleic acid fragment encodes the alpha subunit of the β-conglycinin soybean seed storage protein.
 6. The method of claim 1 wherein at least two members of a class of soybean seed storage protein subunits are reduced when compared to soybeans not containing the chimeric gene of step (a).
 7. A method for simultaneously reducing the expression of two soybean genes comprising: (a) constructing a chimeric gene comprising: (i) a nucleic acid fragment encoding a promoter region from a soybean seed storage protein gene; and (ii) a nucleic acid fragment encoding all or a portion of a soybean protein that is not the soybean seed storage protein of (i), said nucleic acid fragment placed in sense or antisense orientation relative to the promoter of (i), and (iii) a transcriptional termination region; (b) creating a transgenic soybean seed by introducing into a soybean seed the chimeric gene of (a); and (c) growing the transgenic soybean seeds of step (b) under conditions that result in expression of the chimeric gene of step (a) wherein the quantity of one or more members of a class of soybean seed storage protein subunits and the quantity of the protein encoded by the nucleic acid fragment of (a)(ii) is reduced when compared to soybeans not containing the chimeric gene of step (a).
 8. The method of claim 7 wherein the nucleic acid fragment encoding all or a portion of a soybean protein that is not the soybean seed storage protein of (a)(i) is placed in sense orientation relative to the promoter region.
 9. The method of claim 7 wherein the nucleic acid fragment encoding all or a portion of a soybean protein that is not the soybean seed storage protein of (a)(i) is placed in antisense orientation relative to the promoter region.
 10. The method of claim 7 wherein the promoter is derived from the gene encoding the alpha subunit of the β-conglycinin soybean seed storage protein.
 11. The method of claim 7 wherein the nucleic acid fragment encoding all or a portion of a soybean protein that is not the soybean seed storage protein of (a)(i) encodes a gene involved in fatty acid biosynthesis.
 12. The method of claim 7 wherein quantity of one or more members of a class of soybean seed storage protein subunits and the quantity of the protein encoded by the nucleic acid fragment of (a)(ii) are reduced when compared to soybeans not containing the chimeric gene of step (a).
 13. The method of claim 7 wherein the quantity of at least two members of a class of soybean seed storage protein subunits are reduced when compared to soybean seeds not containing the chimeric gene of step (a), and wherein the fatty acid profile of soybean seeds containing the chimeric gene of step (a) is altered when compared to soybean seeds not containing the chimeric gene of step (a).
 14. A transgenic soybean plant prepared by the method of claim
 1. 15. A transgenic soybean plant prepared by the method of claim
 7. 16. Transgenic seeds derived from plants of claim
 14. 17. Transgenic seeds derived from plants of claim
 15. 18. A transgenic soybean plant wherein the quantity of one or more members of a class of soybean seed storage protein subunits is reduced in the seeds of said plant when compared to seeds derived from a non-transgenic soybean plant.
 19. Transgenic seeds derived from plants of claim
 18. 20. A transgenic soybean plant wherein (i) the quantity of one or more members of a class of soybean seed storage protein subunits is reduced; and (ii) the oleic acid content relative to the content of other fatty acids is increased in the seeds of said plant when compared to seeds derived from a non-transgenic soybean plant.
 21. Transgenic seeds derived from plants of claim
 20. 